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Several studies have indicated that both cholesterol and sphingomyelin metabolism can affect the generation of Aβ. In this very elegant paper, Tobias Hartmann’s group has decided to go the opposite way and analyze whether Aβ could affect cholesterol and SM metabolism. They have used several genetic and biochemical approaches to reach the unexpected conclusion that the Aβ peptide can stimulate SM hydrolysis and reduce the biosynthesis of both SM and cholesterol. These effects could potentially be explained by perturbation of the lipid bilayer produced by Aβ. However, the fact that Aβ (in physiological concentrations) can stimulate both a purified neutral SMase (nSMase) activity in vitro and the nSMase activity recovered from cell homogenates suggests a direct effect of the peptide on the enzyme rather than on the lipid environment.

It has long been known that sphingomyelin and cholesterol like to go together (1). Increased biosynthesis of cholesterol is accompanied by increased generation of sphingomyelin. Indeed, the same transcriptional machinery (SREBP) regulates both biosynthetic pathways. The ultimate goal is to keep or cluster cholesterol at the plasma membrane (PM). Sphingomyelin is probably the best “cholesterol-binding lipid” and is highly enriched in the PM. Indeed, its stoichiometry of cholesterol binding is 3:1 (cholesterol:SM), which is extremely high considering that phosphatydylcholine (another common “cholesterol-binding lipid”) binds cholesterol with a 1:1 stiochiometric ratio (cholesterol:PC). This close relationship works the other way around, too. Cell surface hydrolysis of SM is accompanied by a fast translocation of cholesterol to the endoplasmic reticulum (ER). The retro-translocation has the ultimate effect of down-regulating cholesterol biosynthesis (through the HMG-CoA reductase) and increasing the storage of cholesterol ester (which, however, is only temporary and limited to certain cell types). In addition to the effects produced by the retro-translocation of PM-cholesterol, ceramide (one of the products of SM hydrolysis) can down-regulate the proteolysis/activation of SREBP and, therefore, reduce both biosynthesis and uptake of cholesterol (2, 3).

Our group has recently shown that normal aging of the brain is accompanied by activation of nSMase and consequent liberation of the second messenger ceramide, which is able to induce Aβ generation (4). This age-associated effect could be blocked by nSMase inhibitors and by genetic disruption of the ligand-binding domain of the neurotrophin receptor p75NTR, which is responsible for the activation of nSMase (in the brain and during the normal process of aging). If we join the results produced by Grimm et al. and our group (4), we can envision a model in which aging activates ceramide production and Aβ generation by acting through nSMase; Aβ can further stimulate nSMase by an apparent direct interaction, fostering an additional production of Aβ. Sphingomyelin hydrolysis would have the additional effect of reducing cholesterol biosynthesis in astrocytes, affecting the secretion of lipoprotein particles required for neurons to generate/sustain their own synapses (5). In conclusion, a vicious circle might operate that leads to abnormal production of Aβ and affects synaptogenesis. Tobias’s group has shown that the nSMase inhibitor GW4869 can block Aβ production in neurons; our group has shown that a different nSMase, manumycin A, can block Aβ production in both primary neurons and mice (4). This strategy seems to work for both the age-dependent and the Aβ-mediated effect, and is predicted to act upstream of the “vicious circle.”

I have so far considered the effects on cholesterol metabolism described by Grimm et al. as a consequence of SM hydrolysis because there is no evidence of a possible direct effect of the Aβ peptide on the HMG-CoA reductase (at the enzymatic/protein level). Indeed, even though Aβ was given to intact cells, the authors observed a decrease in the incorporation of acetate into the mevalonic pathway; a fact that implicates the HMG-CoA reductase, an ER membrane-based protein. However, we could have yet another surprise and discover that Aβ can act directly on the enzyme itself. It would be interesting to look at SREBP processing and HMG-CoA degradation under the above conditions, and at the effects of Aβ on a purified/enriched preparation of HMG-CoA in vitro.

Finally, one can wonder how the lack of presenilins can stimulate the SM-synthase activity. In fact, in contrast to nSMase, SM-synthase is an allosteric enzyme that seems to respond to the levels of one of its own substrates, palmitoyl-CoA. Interestingly enough, both the mevalonic and the fatty acid/palmitoyl-CoA biosynthetic pathways are under the control of the SREBP family of transcription factors (6). Even though we know that the intramembrane proteolysis of SREBP does not depend on presenilins, I still wonder whether SREBPs play any role behind the curtains. Who knows? Maybe Tobias has another ace ready for us.

We appreciate the interesting study by Hartmann and colleagues. A decade ago we reported that Aβ peptides modulate the cholesterol esterification rate (1). We later showed that Aβ modulates the metabolism of cholesterol and phospholipids (2-4). We studied Aβ's effects on lipid metabolism in a number of test systems, including hepatic cells (2), cultured nerve cells (3), fetal rat brain model (3), and ex vivo in rat hippocampal slices (4) and found that it is tissue and oxidation level-dependent. This is discussed in detail in our recent publication (5) that explored the effects of Aβ on synaptic plasticity and its interrelation with the neural cholesterol homeostasis modulation by Aβ.

Our early study of Aβ's effect on cholesterol esterification was subsequently confirmed by others (6). In this regard, it is important to note that Aβ is a structure-functional component of lipoproteins (7,8,9). Aβ therefore, can affect the reverse cholesterol transport from neuronal tissue to the periphery in addition to its role in cholesterol synthesis and intracellular dynamics. This is supported by earlier studies by Michikawa et al. (10), Igbavboa et al. (11), and us (4), who reported the effects of Aβ on cellular cholesterol uptake and efflux.

"My belief is that Aβ is involved in this interaction by modulating cellular/membrane cholesterol, so, both cholesterol and Aβ (and APP processing) affect each other," I noted three years ago during the ARF live discussion, "Cholesterol and Alzheimer's—Charging Fast but Still at a Distance from Solid Answers." I am glad Dr. Hartmann's skepticism and willingness to see more experiments "to prove this point" has now materialized in the excellent publication by Dr. Hartmann's group.

A wealth of cellular and animal studies indicates that cholesterol regulates Aβ generation. Use of statins is currently being explored as a safe and available strategy that may help protect against Alzheimer disease. While awaiting the outcome of large clinical trials, mechanistic studies are revealing an unexpectedly complex picture of the lipid-Aβ connection. Cholesterol is no longer the only player; cholesteryl-esters, ceramide, sphingomyelin (SM), as well as isoprenoids are among the newest additions to the lipid list. Now, Tobias Hartmann and colleagues add a remarkable twist to the story. Not only do a variety of lipids regulate Aβ generation, but Aβ can also reach back and control cellular cholesterol and SM levels. This provocative conclusion is supported by solid in vitro and in vivo studies, which assign separate functions to Aβ40 (inhibition of HMG-CoA reductase, resulting in decreased cholesterol synthesis) and Aβ42 (activation of SMase, resulting in decreased SM levels). Separate functions of the two peptides are shown in a variety of systems, including in vitro activation of nSMase by Aβ42, but much less by Aβ40; down-regulation of high cellular de novo cholesterol synthesis in APP/APLP2-/- MEF cells by exposure to Aβ40, but not Aβ42; and increased cholesterol together with decreased SM in cells expressing PS1 containing FAD mutations, leading to elevated Aβ42/Aβ40 ratios. Given that cholesterol and SM are integral components of lipid rafts, it would be interesting to examine how lipid raft levels and function are separately regulated by the two peptides in cells expressing FAD mutant presenilins.

This study is important not only for Alzheimer disease, but also for basic cholesterol biology, as Aβ may regulate either HMG-CoA reductase or the SREB pathway. Although Aβ42 appears to directly activate SMase in in vitro assays, the exact mechanism for Aβ40 remains to be elucidated. Exposure of intact cells to Aβ40 reduces the activity of HMG-CoA reductase, an enzyme with established ER localization. The intracellular localization of HMG-CoA reductase would suggest an indirect mechanism of action for exogenous Aβ40. However, extracellular Aβ40 could not normalize cholesterol synthesis in APP/APLP2-/- MEF cells, indicating that perhaps small amounts of intracellular Aβ40 or AICD may also regulate HMG-CoA reductase activity in wild-type cells. Interestingly, lack of Aγ-secretase function in PS1/2-/- MEF cells elevates cholesterol and SM levels quite strongly, while in APP/APLP2-/- MEF cells (which are derived from different mice), levels of both lipids increase more moderately. The implication is that the impact of the γ-secretase/APP/Aβ lipid regulatory system might be quite different in strength depending on which specific cells or tissues are analyzed. One can also ask the question whether, if one looks at other tissues, perhaps there are Aγ-secretase substrates in addition to APP and APLP2 which may regulate cellular cholesterol and SM levels. These and other questions raised by Tobias will further define the delicate network of the newly established reciprocal lipid-Aβ connection.